US11198864B2ActiveUtilityA1

Protease-resistant systems for polypeptide display and methods of making and using thereof

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Assignee: CYTOMX THERAPEUTICS INCPriority: Aug 10, 2012Filed: Mar 2, 2020Granted: Dec 14, 2021
Est. expiryAug 10, 2032(~6.1 yrs left)· nominal 20-yr term from priority
Inventors:Sherry Lynn La PorteStephen MooreJames William West
C12N 15/1037C40B 30/06C40B 40/02G01N 33/6845C07K 14/705C12Q 1/37C07K 16/2809C07K 14/001C07K 16/18C07K 2319/02C12N 15/1044C07K 16/2866C07K 2319/03C07K 2317/622C07K 2319/21
74
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Cited by
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References
34
Claims

Abstract

The present invention generally relates to bacterial polypeptide display systems, libraries using these bacterial display systems, and methods of making and using these systems, including methods for improved display of polypeptides on the extracellular surface of bacteria using circularly permuted transmembrane bacterial polypeptides that have been modified to increase resistance to protease degradation and to enhance polypeptide display characteristics.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
       1. A polypeptide display library comprising:
 a plurality of replicable biological entities, each member of the plurality comprising a displayed moiety (DM) displayed on the surface of the member replicable biological entity, wherein the DM is fused to an extracellular terminus of a circularly permuted bacterial outer membrane carrier protein (CP) to produce a display platform (DP), 
 wherein the CP comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 56 and SEQ ID NO: 57. 
 
     
     
       2. The polypeptide display library of  claim 1 , wherein the DP comprises a tag. 
     
     
       3. The polypeptide display library of  claim 2 , wherein the tag is a fluorescent agent. 
     
     
       4. The polypeptide display library of  claim 2 , wherein the tag is a C-terminal tag. 
     
     
       5. The polypeptide display library of  claim 2 , wherein the tag is an N-terminal tag. 
     
     
       6. The polypeptide display library of  claim 1 , wherein the replicable biological entity is a bacterial cell. 
     
     
       7. The polypeptide display library of  claim 1 , wherein the CP comprises a glycine-serine polypeptide sequence N-terminal to the amino acid sequence of the CP, C-terminal to the amino acid sequence of the CP, or both N-terminal to and C-terminal to the amino acid sequence of the CP. 
     
     
       8. The polypeptide display library of  claim 7 , wherein the glycine-serine polypeptide sequence N-terminal to the amino acid sequence of the CP comprises an amino acid sequence selected from the group consisting of: GQSGQ (SEQ ID NO: 7), GGSGQSGQGG (SEQ ID NO: 9), GGSGQSGQ (SEQ ID NO: 22), GGSGQSGQGS (SEQ ID NO: 59), GGSG (SEQ ID NO: 15), GGSGGSGGSGGSG (SEQ ID NO: 20), SGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 24), and GSSGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 11). 
     
     
       9. The polypeptide display library of  claim 7 , wherein the glycine-serine polypeptide sequence C-terminal to the amino acid sequence of the CP comprises an amino acid sequence selected from the group consisting of: GGS (SEQ ID NO: 17), GGSGGSSGQAAAG (SEQ ID NO: 12), and GGSGGSSGQTAAG (SEQ ID NO: 83). 
     
     
       10. The polypeptide display library of  claim 7 , wherein
 the glycine-serine polypeptide sequence N-terminal to the amino acid sequence of the CP comprises an amino acid sequence selected from the group consisting of: GQSGQ (SEQ ID NO: 7), GGSGQSGQGG (SEQ ID NO: 9), GGSGQSGQ (SEQ ID NO: 22), GGSGQSGQGS (SEQ ID NO: 59), GGSG (SEQ ID NO: 15), GGSGGSGGSGGSG (SEQ ID NO: 20), SGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 24), and GSSGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 11), and 
 the glycine-serine polypeptide sequence C-terminal to the amino acid sequence of the CP comprises an amino acid sequence selected from the group consisting of: GGS (SEQ ID NO: 17), GGSGGSSGQAAAG (SEQ ID NO: 12), and GGSGGSSGQTAAG (SEQ ID NO: 83). 
 
     
     
       11. A polypeptide display library comprising:
 a plurality of replicable biological entities, each member of the plurality comprising a display platform (DP) comprising a carrier polypeptide (CP) operably linked to a displayed moiety (DM), 
 wherein the CP comprises a circularly permuted barrel-shaped beta sheet transmembrane polypeptide and a glycine-serine polypeptide sequence N-terminal to the amino acid sequence of the CP, C-terminal to the amino acid sequence of the CP or both N-terminal to and C-terminal to the amino acid sequence of the CP, and 
 wherein the transmembrane polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 56 and SEQ ID NO: 57. 
 
     
     
       12. The polypeptide display library of  claim 11 , wherein the DM is displayed N-terminal to the amino acid sequence of the CP. 
     
     
       13. The polypeptide display library of  claim 11 , wherein the DM is displayed C-terminal to the amino acid sequence of the CP. 
     
     
       14. The polypeptide display library of  claim 11 , wherein the glycine-serine polypeptide sequence N-terminal to the amino acid sequence of the CP comprises an amino acid sequence selected from the group consisting of GQSGQ (SEQ ID NO: 7), GGSGQSGQGG (SEQ ID NO: 9), GGSGQSGQ (SEQ ID NO: 22), GGSGQSGQGS (SEQ ID NO: 59), GGSG (SEQ ID NO: 15), GGSGGSGGSGGSG (SEQ ID NO: 20), SGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 24), and GSSGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 11). 
     
     
       15. The polypeptide display library of  claim 11 , wherein the glycine-serine polypeptide sequence C-terminal to the amino acid sequence of the CP comprises an amino acid sequence selected from the group consisting of GGS (SEQ ID NO: 17), GGSGGSSGQAAAG (SEQ ID NO: 12), and GGSGGSSGQTAAG (SEQ ID NO: 83). 
     
     
       16. The polypeptide display library of  claim 11 , wherein the CP comprises a glycine-serine polypeptide sequence both N-terminal to and C-terminal to the amino acid sequence of the CP. 
     
     
       17. The polypeptide display library of  claim 16 , wherein the glycine-serine polypeptide sequence N-terminal to the amino acid sequence of the CP comprises an amino acid sequence selected from the group consisting of GQSGQ (SEQ ID NO: 7), GGSGQSGQGG (SEQ ID NO: 9), GGSGQSGQ (SEQ ID NO: 22), GGSGQSGQGS (SEQ ID NO: 59), GGSG (SEQ ID NO: 15), GGSGGSGGSGGSG (SEQ ID NO: 20), SGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 24), and GSSGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 11), and the glycine-serine polypeptide sequence C-terminal to the amino acid sequence of the CP comprises an amino acid sequence selected from the group consisting of GGS (SEQ ID NO: 17), GGSGGSSGQAAAG (SEQ ID NO: 12), and GGSGGSSGQTAAG (SEQ ID NO: 83). 
     
     
       18. The polypeptide display library of  claim 11 , wherein the DP comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 58, SEQ ID NO: 81, and SEQ ID NO: 82. 
     
     
       19. The polypeptide display library of  claim 11 , wherein the DP comprises a tag N-terminal to the amino acid sequence of the CP, a tag C-terminal to the amino acid sequence of the CP, or both a tag N-terminal to the amino acid sequence of the CP and a tag C-terminal to the amino acid sequence of the CP. 
     
     
       20. The polypeptide display library of  claim 19 , wherein the DP has the structural arrangement from N-terminus to C-terminus: T1-CP-DM-T2 or T1-DM-CP-T2, wherein T1 and T2 are tags. 
     
     
       21. The polypeptide display library of  claim 19 , wherein the tag N-terminal to the amino acid sequence of the CP comprises the amino acid sequence EYMPME (SEQ ID NO: 8). 
     
     
       22. The polypeptide display library of  claim 19 , wherein the tag C-terminal to the amino acid sequence of the CP comprises a histidine tag. 
     
     
       23. The polypeptide display library of  claim 22 , wherein the histidine tag is an 8-His tag. 
     
     
       24. The polypeptide display library of  claim 19 , wherein the tag N-terminal to the amino acid sequence of the CP comprises the amino acid sequence EYMPME (SEQ ID NO: 8) and the tag C-terminal to the amino acid sequence of the CP comprises an 8-His tag. 
     
     
       25. The polypeptide display library of  claim 19 , wherein the DP comprises one or more linkers. 
     
     
       26. The polypeptide display library of  claim 25 , wherein the DP comprises a linker between the tag N-terminal to the amino acid sequence of the CP and the DM. 
     
     
       27. The polypeptide display library of  claim 25 , wherein the DP comprises a linker between the DM and the transmembrane polypeptide. 
     
     
       28. The polypeptide display library of  claim 25 , wherein the DP comprises a linker between the transmembrane polypeptide and the tag C-terminal to the amino acid sequence of the CP. 
     
     
       29. The polypeptide display library of  claim 25 , wherein the DP comprises a linker between the tag N-terminal to the amino acid sequence of the CP and the DM, a linker between the DM and the transmembrane polypeptide, and a linker between the transmembrane polypeptide and the tag C-terminal to the amino acid sequence of the CP. 
     
     
       30. The polypeptide display library of  claim 11 , wherein the DP comprises more than one DM. 
     
     
       31. The polypeptide display library of  claim 30 , wherein the DP has the structural arrangement from N-terminus to C-terminus: T1-DM1-DM2-CP-T2, T1-CP-DM1-DM2-T2, T1-DM1-DM2-DM3-CP-T2, T1-CP-DM1-DM2-DM3-T2, DM1-T1-DM2-CP-T2, T1-CP-DM1-T2-DM2, or T1-DM1-CP-DM2-T2, wherein T1 and T2 are tags. 
     
     
       32. The polypeptide display library of  claim 11 , wherein the DP comprises one or more linkers. 
     
     
       33. The polypeptide display library of  claim 11 , wherein the DM is selected from the group consisting of: a substrate, an exosite, a masking moiety, an antibody, a receptor, a ligand, and other proteins, and any combinations thereof. 
     
     
       34. The polypeptide display library of  claim 33 , wherein the DM is a substrate, and wherein the substrate is a substrate for a protease.

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